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A.-J. F. Carr, A. A. Vugler, S. E. Moss, P. J. Coffey, J. Greenwood; Transdifferentiation of the ARPE-19 Cell Line Towards a Photoreceptor Phenotype. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1933.
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The synthetic retinoid agonist, fenretinide, induces a neuronal-like phenotype in the spontaneously immortalized human cell line ARPE-19. This differentiation is characterized by the formation of neural processes, and is accompanied by the expression of microfilaments 160 and 200, and calretinin, markers not normally associated with RPE cells (Chen, 2003). Here we have assessed the potential of fenretinide to induce the formation of photoreceptor cells from ARPE-19 cells.
ARPE-19 cells were cultured in standard medium (DMEM:F12 + Glutamax, 10% FCS and 1x pen/strep) prior to the experiment. Cells were passaged on Day 1 and plated at a concentration of 2.2x103 cells/cm2 in standard medium. On Day 2 the medium was replaced with experimental medium (DMEM:F12 + Glutamax, 3% charcoal-dextran treated FBS, 1x pen/strep). The following day, cells were washed with 1xHBSS and treated with experimental medium containing fenretinide, solubilised in DMSO, at as final concentration of 3µM. Control cells were treated with experimental medium containing the same volume of DMSO only. This treatment was repeated each day for 7 days in total. Cells were fixed and processed for immunocytochemistry, lysed for western blotting and collected in TRIsol for RNA analysis. The expression of the photoreceptor cell markers was then examined.
Fenretinide treated ARPE-19 cells exhibited dramatic morphological changes, evident by the formation of neuronal processes and neurite branching. The expression of rhodopsin, M/L opsin and recoverin was increased in fenretinide-treated cells, as compared to DMSO-treated control cells. Furthermore, the expression of cytokeratin 8, an RPE cell marker, was decreased in fenretinide treated cells.
The upregulation of rhodopsin, M/L opsin and recoverin, together with the down regulation of cytokeratin8 in fenretinide-treated ARPE-19 cells suggests that ARPE-19 cells possess the capacity to transdifferentiate into photoreceptor cells.
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